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THE    MECHANISM    OF   NATURAL    AND 
ACQUIRED  STREPTOCOCCUS  IMMUNITY 


A  DISSERTATION 

SUBMITTED  TO  THE  FACULTY  OF 

OGDEN  GRADUATE  SCHOOL  OF  SCIENCE 

IV    ('AXIHDACY    FOR    THE 
DEGREE  OI'1 

DOCTOR  OF  PHILOSOPHY 


(DEPARTMENT  OF  PATHOLOGY   AND  BACTERIOLOGY 


GUSTAV  F.  RUEDIGER 


THni\>ersits  of  Cbtcago 

FOUNDED  BV  JOHN  D.  ROCKEFELLER 


THE  MECHANISM  OF  NATURAL  AND 

ACQUIRED  STREPTOCOCCUS 

IMMUNITY 


A  DISSERTATION 

SUBMITTED  TO  THE  FACULTY  OF  THE  OGDEN    GRADUATE    SCHOOL    OF 

SCIENCE    IN    CANDIDACY    FOR    THE    DEGREE    OF 

DOCTOR    OF    PHILOSOPHY 

(DEPARTMENT  OF  PATHOLOGY  AND  BACTERIOLOGY) 


BY 

GUSTAV  F.  RUEDIGER 
h 


' 


CHICAGO 
1907 


(From  the  Memorial  Institute  for  Infectious  Diseases,  Chicago.) 


THE  MECHANISM  OF  NATURAL 
AND  ACQUIRED  STREPTOCOCCUS  IMMUNITY. 

EY 

GUSTAV  F.  RUEDIGFR. 

PROFESSOR     OF  BACTERIOLOGY     AND     PATHOLOGY,      UNIVERSITY     OF      NORTH 
DAKOTA,  GRAND  FORKS,  N.  D. 

INTRODUCTION. 

A  great  deal  of  work  has  been  done  on  the  mechanism  of  strepto- 
coccus immunity  both  with  normal  and  with  immunized  animals,  but 
many  points  are  still  left  entirely  in  the  dark.  In  the  experiments  de- 
scribed in  this  paper,  an  attempt  is  made  to  throw  additional  light  on 
some  of  these  points,  but  on  account  of  the  many  inherent  difficulties,, 
the  results  which  I  have  achieved  by  no  means  exhaust  the  problem. 

Shortly  after  the  publication  of  his  classical  paper  on  phagocytosis 
in  daphnia,  MetchnikofP  made  a  study  of  phagocytosis  of  streptococci: 
in  the  human  body,  in  cases  of  erysipelas.  He  concluded  from  his  ob- 
servations that  streptococci  gain  entrance  through  abrasions  of  the  skin,, 
multiply  and  set  up  an  inflammation.  At  the  same  time  there  is  a  gath- 
ering of  microcytes  about  the  streptococci,  and  these  take  up  the  latter 
and  destroy  them.  Macrocytes  are  also  found  in  considerable  numbers, 
but  they  do  not  take  up  the  streptococci,  but  have,  nevertheless,  a  phago- 
cyte action  in  that  they  take  up  and  remove  dead  and  disabled  micro- 
cytes. 

In  1895  Denys  and  Leclef*  studied  the  mechanism  of  immunity  in 
rabbits  which  had  been  repeatedly  injected  with  small  but  gradually  in- 
creased doses  of  virulent  streptococci.  Their  conclusions  may  be  briefly 
stated  as  follows :  Immune  rabbit  serum  is  not  so  good  a  culture  medium 
for  streptococci  as  normal  serum  but  it  does  not  possess  any  marked, 
streptococcidal  powers.  The  cell-free  fluid  from  a  leucocytic  exudate 

*Virdiow's  Archiv.  1887,  evil.,  209. 
**La  Cellule,  1805,  XI,  177. 

183473 


retards  multiplication  of  virulent  streptococci  and  sometimes  kills  them. 
Normal  leucocytes  in  normal  serum,  and  leucocytes  from  an  immune 
rabbit  suspended  in  normal  serum,  do  not  greatly  retard  multiplication 
of  virulent  streptococci.  Leucocytes  from  a  normal  rabbit,  or  those  from 
an  immune  rabbit,  suspended  in  immune  rabbit  serum  rapidly  take  up 
and  destroy  virulent  streptococci.  The  serum  has  acquired  something 
in  the  process  of  immunization  \vhich  neutralized  something  in  the  cocci 
by  virtue  of  which  they  were  protected  against  phagocytosis. 

Denys  and  Marchand*  showed  that  there  is  better  phagocytosis  ot 
virulent  streptococci  when  inoculated  into  a  suspension  of  rabbit's  leu- 
cocytes in  normal  rabbit  serum  to  which  had  been  added  I  per  cent,  of 
immune  horse  serum,  than  when  they  were  inoculated  into  the  same 
mixture  of  leucocytes  and  rabbit  serum  with  the  addition  of  I  per  cent, 
of  normal  horse  serum. 

Bordet*  was  able  to  find  no  protection  against  streptococci  in  normal 
and  immunized  rabbits  and  guinea-pigs  except  that  due  to  phagocy- 
tosis. If  a  rabbit  was  treated  with  antistreptococcic  serum,  and  later 
injected  with  several  times  the  minimum  fatal  dose  of  streptococci,  the 
organisms  were  all  taken  up  by  phogacytes  and  destroyed.  In  untreated 
rabbits  he  also  found  phagocytosis,  but  the  organisms  soon  got  the  upper 
hand  and  the  rabbit  died  of  streptococcus  infection. 

Marchand*  studied  phagocytosis  of  virulent  and  non-virulent  strep- 
tococci, and  came  to  the  conclusion  that  the  failure  on  the  part  of  the 
leucocytes  to  take  up  virulent  streptococci  depends  on  a  physical  property 
of  the  organisms  and  not  on  a  secretion.  Tchistovitch**  injected  rab- 
bits intravenously  with  fatal  doses  of  a  highly  virulent  streptococcus, 
killed  the  animals  in  one-fourth  to  six  hours  and  examined  the  organs 
for  evidence  of  phagocytosis.  He  was  always  able  to  find  some  phagocy- 
tosis in  the  lungs  and  in  the  livers  of  these  animals,  and  concluded  that 
this  phagocytosis  of  virulent  streptococci  may  be  due  to  the  fact  that 
every  culture  contained  some  cocci  which  are  less  resistant  than  others, 
and  that  those  are  the  only  ones  which  are  taken  up  by  the  phagocytes. 

Simon*  found  very  little  evidence  of  phagocytosis,  in  vitro,  when 
rabbit's  leucocytic  exudate  was  inoculated  with  a  non-virulent  strepto- 
coccus. The  washed  leucocytes  suspended  in  salt  solution,  and  also  the 
cell-free  exudate,  killed  non-virulent  streptococci,  but  not  the  virulent 
organisms.  Leucocytic  exudate  mixed  with  rabbit  serum  had  no  effect 

•Quoted  from  Donys,  (Vntralbl.  f.  Bact,  1898,  XXIV,  685. 
**Aun.  tie  I'lhst.  J'Mstour.  ISiMi.  X,  104,  and  18!)7,  XI,  177. 
•Archiv.  do  M('<].   K.\»><>r..    ISDN.  x.  r>r>:j. 
**An.  de  1'Inst.  Pasteur.  IfXiO.  XIV,  802. 
*Ontnilbl.  f.  liMkt.  V.xn.  XXIX.  SI  and  li:',. 


on  non -virulent  streptococci.  He  .was  unable  to  confirm  the  view  ad- 
vanced by  Bordet  and  others  that  a  rabbit  which  is  injected  with  strep- 
tococci into  a  pleural  cavity  containing  leucocytic  exudate  can  stand  a 
larger  dose  than  a  rabbit  of  the  same  size  which  is  injected  in  the  normal 
pleura.  The  pleural  exudate  was  produced  by  the  injection  of  aleuronat 
suspension  forty-eight  hours  previously.  These  results  are  quite 
contradictory  to  those  of  other  investigators,  and  it  was,  therefore, 
thought  desirable  to  repeat  these  experiments  with  leucocytes. 

Neufeld  and  Rimpatt*  have  shown  that  the  leucocytes  play  an  im- 
portant role  in  combating  streptococcus  infections  in  immunized  rabbits. 
They  have  shown  also  that  the  addition  of  antistreptococcus  serum  to  a 
suspension  of  leucocytes  and  streptococci  aids  phagocytosis  and  that  this 
is  not  due  to  a  stimulation  of  the  phagocytes  but  to  an  effect  of  the  serum 
•on  the  cocci.  The  specific  substance  in  the  immune  serum  they  found 
to  be  fairly  resistant  to  heat,  it  being  unchanged  by  heating  at  59°  C.  for 
^2  hour. 

TEST  TUBE   EXPERIMENTS   WITH   LEUCOCYTIC  EXUDATE  FROM    NORMAL  ANI- 
MALS. 

The  leucocytic  exudate  used  in  these  experiments  was  obtained 
by  injecting  a  6-8%  suspension  of  aleuronat  in  Na  Cl  solution  into  the 
right  pleural  cavity  of  a  rabbit  or  large  guinea-pig  and  bleeding  the 
animal  to  death  eight  to  ten  hours  later.  The  aleuronat  suspension  is 
best  prepared  by  sterilizing  the  aleuronat  in  dry  heat  at  150°  to  160°  C. 
and  suspending  it  in  sterile  physiological  salt  solution.  No  sodium 
oxalate  was  added  to  the  exudate,  as  this  salt  is  strongly  streptococcidal, 
but  the  coagulation  of  the  exudate  was  prevented  by  occasional  agitation 
of  the  tubes  during  the  first  hour.  The  contents  of  each  tube  were  2  c.  c., 
which  were  inoculated  with  three  loopfuls  of  a  twenty-four-hour  broth 
streptococcus  culture  and  three  loopfuls  were  plated  at  intervals.  Tube 
7  differs  from  Tube  6,  in  that  it  contains  the  washed  leucocytes  which 
had  been  centrifugated  out  of  2  c.  c.  of  the  exudate,  suspended  in  rabbit 
serum,  whereas  Tube  6  contained  a  mixture  of  I  c.  c.  of  the  entire  exu- 
date -f-  i  c.  c.  of  serum.  The  fluid  of  the  exudate  in  Tube  7  was  removed 
to  show  that  the  effect  of  these  mixtures  is  not  due  entirely  to  the  liquid 
portion  of  the  exudate,  but  in  a  large  measure  to  the  leucocytes.  The 
Tables  show  that  non-virulent  streptococci  are  destroyed  by  the  leuco- 
cytic exudate,  by  mixtures  of  leucocytes  and  serum  and  by  leucocytes  in 
clefibrinated  blood.  Defibrinated  blood  and  serum  alone,  the  heated  exti- 

**D.  Med.  Wool].,  11)04,  XXX,  1458. 


date  and  a  suspension  of  leucocytes  in  salt  solution  or  in  heated  serum- 
have  little  or  no  effect  on  these  organisms.  The  virulent  streptococcus 
is  not  destroyed  by  the  leucocytic  exudate  nor  by  a  suspension  of  leuco- 
cytes in  serum  or  blood.  Smears  made  in  one  to  two  hours  from  tubes 
in  which  there  is  destruction  of  cocci  show  evidence  of  phagocytosis. 
Table  2  shows  the  same  experiment  as  that  shown  in  Table  I ;  except  that 
guinea-pig's  leucocytes  and  blood  were  used.  With  these  leucocytes  the 

TABLE  1. 

SHOWING   THE  EFFECT   OF   RABBIT'S   LEUCOCYTIC   EX  L- DATE   AND    MIXTURES    OF   EXl  DATE 
AXD  BODY  FLUIDS  OX  STUEI'TOCOCCI. 

Strepto-  Immecli- 

eocci.  ately.  3  hrs.  18  hrs. 

300     Leucocytic  exudate 4GOO  0  10000 

300     Exudate  58°  for  half  hour 3250  8000  Many 

300    Defibrinated  blood 4800  4700  Many 

300    Blood  1  c.  c.  +  exudate  1  c.  c 4000  750  1 500 

300    Rabbit  serum 4200  4200  Many 

300     Serum  1  c.  c.  -f  exudate  1  c.  e 5200  1100  Many 

300    Leucocytes  -f  serum 5000  800  Many 

300    Leucocytes  in  heated  serum 5500  3560  Many 

300    Leucocytes  in  NaCl  sol 4900  5000  Many 

300    Leucocytes  in  NaCl  sol.  -f  Sensitized  streptococci 570  32  .",000 

324    Leucocyte  exudate  420  11  Many 

324    Defibrinated  blood  430  700O  Many 

324     Blood  1  c.  c.  -f-  exudate  1  c.  c 750  540  Many 

324    Rabbit  serum    552  0500  14000 

324     Serum  1  c.  c.  +  exudate  1  c.  c 320  1  Many 

B104    Leucocytic  exudate 3500  3600  Many 

270    Leucocytic  exudate   1700  2  Many 

Sources  of  streptococci:  Nos.  300  and  324  from  the  heart's  blood  of  scarlet 
fever  patients,  postmortem.  No.  B104  from  an  abscess  in  a  guinea-pig  which 
had  been  injected  with  a  fungus.  This  organism  has  been  passed  through  78 
rabbits  and  is  very  virulent  for  both  rabbits  and  guinea-pigs. 

eighteen-hour  plates  are  often  sterile,  or  nearly  so,  while  it  is  only  occa- 
sionally that  they  are  sterile  when  rabbit's  leucocytes  are  used. 

Special  attention  must  be  called  to  the  fact  that  the  suspension  of 
leucocytes  in  salt  solution  destroys  many  of  the  sensitized  a  virulent  strep- 
tococci. These  results  are  therefore  in  harmony  with  the  work  of 
Wright  and  Douglas/-'  who  have  shown  that  phagocytosis  takes  place  only 
after  the  bacteria  have  been  sensitized,  that  is,  have  been  acted  on  by 
the  opsonin  of  the  serum.  There  is  no  phagocytosis  in  a  suspension  of 
washed  leucocytes  in  salt  solution  or  in  heated  serum,  when  untreated 

*Proc.  of  Royal  'Soc.,  1903,  LXXJI,  357,  and  J904,  LXXIII,  128. 


5 

Bacteria  are,  added,  but  when  sensitized  bacteria  are  added  to  such  a 
suspension  of  leucocytes  there  is  good  phagocytosis.  This  work  was 
confirmed  and  extended  by  Hektoen  &  Ruediger**  and  by  Bulloch  & 
Atkin.***  In  consideration  of  these  facts  we  would  expect  that  the 

TABLE   2. 

-SHOWING    THE    EFFECT    OF    GUINEA-PIG*S    I.EUCOCYTIC    EXUDATE    AND    MIXTURE    OF    EXU- 
DATi:   AND  BODY   FLUIDS'  ON    STREPTOCOCCI. 

Strepto-  Immedi- 

•cocci.  ately.  3  hrs.  18  hrs. 

300  Leucocytic  exndate  6500  70  170 

A  Leucocytic  exndate  650  600  Many 

B104  Leucocytic  exudate  8000  10000  Many 

300  Cell-free  exudate 5500  800  GOflO 

7H04  Cell-free  exudate 8000  10000  Many 

300  Defibrinated  blood .6000  5000  Many 

300  Defibrinated  blood  +  leucocytes  6800  240  72 

300  Serum  6000  6000  Many 

300  Serum  +  leucocytes 6000  2000  Many 

Streptococcus  Xo.  300  is  not  virulent  for  guinea-pigs.  Nos.  B104  and  A  are 
•virulent.  No.  A  was  isolated  from  a  peritonsillar  abscess  following  an  attack 
of  follicular  tonsillitis  and  was  used  in  this  experiment  in  the  first  generation 
on  artificial  media. 

TABLE  3. 

PHAGOCYTOSIS    OF    STREPTOCOCCI    BY   RABBIT    LEUCOCYTES    AND   GUINEA-PIG    LEUCOCYTES. 

Strepto-  Phagocy- 

cocci.  tosis. 

300  Rabbit  leucocytes  in  blood    20 

B104  Rabbit  leucocyte?   in   blood : .  0 

300  Guinea-pig  leucocytes  in  blood 30 

13104  Guinea-pig  leucocytes  in  blood  1 

300  Washed  rabbit  leucocytes  in  NaCl  solution 1 

300  Washed  rabbit  leucocytes  in  heated  rabbit  serum 1.5 

300  Washed  rabbit   leucocytes   -f  sensitized  streptococci   12 

Each  tube  contained  2  c.  c.  of  suspension  of  leucocytes  -(-  2  c.  c.  of  suspen- 
sion of  streptococci.  Tubes  were  incubated  1  hour,  smears  were  made  and 
stained  and  the  degree  of  phagocytosis  determined  by  counts. 

extent  of  destruction  of  streptococci  in  the  test  tubes  should  run  parallel 
with  the  degree  of  phagocytosis  of  these  organisms  and  Table  3  shows 
that  this  is  the  case.  0* ' 

If  we  read  the  three  tables  together  we  notice  that  the  virulent 

**Jour.  of  Infect.  Dis.,  1905,  II,  128. 
***Proc.  of.  Royal  Soc.,  1905,  LXXIV,  379. 


organism  (6104)  is  not  taken  up  by  the  rabbit  leucocytes  and  guinea-pig^ 
leucocytes  in  blood,  neither  is  this  organism  destroyed  or  killed  by  the 
leucocytic  exudate  and  suspensions  of  leucocytes  from  these  animals  in 
blood  or  fresh  serum.  The  avirulent  organism  (300)  is  ingested  by  the 
leucocytes  in  blood  and  in  serum  but  not  by  washed  leucocytes  in  Na  Q 
solution  or  in  heated  serum  unless  the  cocci  have  been  treated  before- 
hand with  fresh  unheated  serum  (sensitized).  Similarly  this  organism 
is  destroyed  by  suspensions  of  leucocytes  in  blood  or  serum  but  not  by 
suspension  of  washed  leucocytes  in  NaCl  solution  or  heated  serum.  The- 
washed  leucocytes  do  however  destroy  the  sensitized  avirulent  organisms. 

EXPERIMENTS   WITH   ORGAN    CELLS. 

It  seems  quite  reasonable  to  suppose  that  the  organ  cells  might  play 
an  important  part  in  the  protection  of  the  body  against  invasion  by 
streptococci ;  and  with  this  point  in  mind  the  following  experiment  was 
carried  out: 

Experiment. — Two  guinea-pigs,  A  and  B,  were  bled  to  death  and' 
the  blood  collected  under  aseptic  precautions.  The  organs  were  now  care- 
fully removed  from  the  body,  placed  in  sterile  glass  dishes  and  rubbed 
up:  (A)  with  guinea-pig's  serum,  and  (B)  with  defibrinated  guinea- 
pig's  blood.  The  organs  and  blood  were  not  allowed  to  become  cold. 

TABLE  4. 

SHOWING    THE    EFFECT    OF    THF    SUSPENSIONS    OF     ORGAN    CELLS    OF    GriNEA-PK.S     ON 

STREPTOCOCCI. 

Strepto-  Iinmedi- 

cocci.  ately.  •'»  lirs.  IN  hrs. 

B104  -+-  Defibrinated  blood 1800  HHM •  1  500- 

B104         Blood    +    muscle    2000  8000  Many 

B104         Blood  -I-  spleen 1900  Many  Many 

B104         Blood  +  liver   1800  Many  Many 

B104         Blood  -f-  kidney 1900  Many  Many 

B104         Blood   +Hdrena1    1500  Many  Many 

B104        Blood  +  lymph  gland  1500  0000  Ma  ny 

300         Blood  -f  bone  marrow  2500  lOoo  Many 

300        Serum 3SOO  :;75o  (5000 

300        Serum  -j-  leucocytes 2700  1800  1500 

300         Serum  +  spleen 2800  50OO  Many 

300         Serum  -j-  liver 2000  12000  Many 

300        Serum  +  kidney 2400  12000  Many 

Control  liver  +  serum 0  •"•  !> 

Control  ppleeu  +  serum 0  0  0- 

The  strain  of  streptococcus  B104  used  in  this  experiment  has  not  been  passed 
through  rabbits  and  has  lost  its  virulence. 


The  suspensions  of  organ  cells  were  now  introduced  into  small  test  tubc.% 
inoculated  with  two  loopfuls  of  a  24-hour  broth  culture  of  a  non-virulent 
streptococcus,  and  two  loopfuls  were  plated  at  intervals.  The  colonies 
that  developed  in  each  plate  were  carefully  estimated  and  the  results 
are  shown  in  Table  4.  In  no  instance  did  the  suspension  of  organ  cells 
have  an  inhibitive  effect  on  the  multiplication  of  the  streptococci,  but 
on  the  contrary  the  organisms  multiplied  more  rapidly  in  these  suspen- 
sions than  in  either  the  serum  or  the  defibrinated  blood  alone.  If,  how- 
ever, bone  marrow  was  added  to  the  defibrinated  blood  or  serum,  the 
number  of  streptococci  in  these  tubes  was  greatly  diminished  during  the 
first  three  to  five  hours. 

These  results  agree  with  those  found  by  Bail  and  Pettersson*  in 
their  study  of  the  mechanism  of  anthrax  immunity  in  chickens,  and  show 
that  suspensions  of  organ  cells  from  the  guinea-pig  possess  no  strepto- 
coccidal  power,  except  the  suspensions  of  bone  marrow.  Suspensions 
of  marrow  cells  in  defibrinated  blood  or  serum  kill  large  numbers  of 
avirulent  streptococci,  but  that  is  not  the  case  with  suspensions  in  salt 
solution.  These  results  have  also  been  obtained  with  suspensions  of 
rabbit's  bone  marrow. 

PHAGOCYTOSIS  OF  LIVING  STREPTOCOCCI   IN  VIVO. 

It  having  been  found  that  leucocytes  ingest  and  destroy  streptococci 
in  test  tube  experiments,  the  following  experiment  was  made  to  show 
that  there  is  phagocytosis  of  living  streptococci  in  the  animal  body: 

A  guinea-pig  was  injected  in  the  right  pleura  with  7  c.  c.  of 
aleuronat  suspension.  Twelve  hours  later  he  was  injected  in  tin's 
pleura  with  2  c.  c.  of  an  1 8-hour  glucose  broth  culture  of  a  non- 
virulent  streptococcus,  and  two  hours  after  this  injection  he  was  bled  to 
death.  Several  cubic  centimeters  of  leucocytic  exudate  were  now  taken 
from  the  right  pleura,  under  aseptic  precautions.  Smears  made  with 
this  exudate  contained  no  free  streptococci,  but  many  of  the  leucocytes 
contained  cocci  in  large  numbers. 

Figure  i  is  a  photomicrograph  of  one  of  these  smears  showing 
streptococci  in  the  body  of  a  leucocyte. 

Preparation  of  Slides. — By  means  of  a  sterile,  soft  cotton  swab  a 
small  quantity  of  the  exudate  was  now  gently  smeared  on  each  of  eight 
agar-covered  slides ;  the  slides  were  incubated  in  a  moist  chamber  for 
four  to  six  hours,  stained  and  examined  under  the  microscope.  The  prep- 
aration of  these  slides  requires  the  following  special  technic.  Three 
ordinary  clean  glass  slides  and  a  heavy  watch  glass  are  placed  in  a  large 

*Centnilbl.  f.  Bakt,  1903,  XXXV,  102. 


8 

Petri  dish  and  sterilized  in  dry  heat.  When  the  Petri  dish  has  cooled 
.sufficiently  to  be  handled  the  slides  are  taken  out  with  sterile  forceps  and 
hot  glucose  agar  is  poured  on  each  to  form  a  uniform  thin  film.  Each 
.slide  is  immediately  replaced  in  the  Petri  dish  and  the  agar  allowed  to 
solidify.  The  exudate  which  contains  many  streptococci  in  the  bodies 
of  the  leucocytes  is  now  gently  smeared  on  these  agar  films,  the  watch 
.glass  is  half  filled  with  sterile  water,  and  the  large  dish  is  placed  in 
the  incubator  for  four  to  six  hours.  The  water  is  used  to  prevent  dry- 
ing of  the  agar. 

Staining. — The  slides  are  now  taken  out  of  the  Petri  dish  and  dried 


Fig.   1. — Photomicrograph   x   1300.     Streptococci  in  guinea-pig's  leucocytes, 
taken  from  the  pleura  t\vo  hours  after  injection  of  streptococci. 


in  a  thermostat  at  55  C.  for  fifteen  to  twenty  minutes,  or  longer  if  nec- 
essary; fixed  in  40  per  cent,  formalin  for  four  or  five  minutes,  washed 
in  water,  stained  for  only  a  few  seconds  with  Loeffler's  methylene  blue 
solution,  again  washed  in  water  and  dried  in  the  thermostat. 

The  best  stains  were  obtained  when  the  methylene  blue  solution  was 
poured  over  the  wet  slide,  the  slide  being  held  so  that  the  stain  ran  off 
readily.  The  slide  is  then  immediately  placed  in  a  dish  of  cold  water 
and  gently  rinsed  in  several  changes  of  water.  Violent  agitation  in  the 
water  pulls  off  the  film  of  agar.  When  the  agar  is  perfectly  dry  (which 
requires  only  about  half  an  hour),  a  cover-glass  is  placed  on  it,  either 


9 

in  glycerin  or  Canada  balsam.    The  slides  are  now  ready  to  be  examined 
with  the  oil  immersion  lens. 

Examination. — Examination  in  various  stages  shows  that  the  strep- 
tococci in  the  leucocytes  multiply,  burst  open  the  cell  and  form  a  small 
colony  surrounding  the  cell  which  contained  them.  Figures  2  and  3  are 
photomicrographs  of  such  preparations  at  different  stages.  In  Figure  2 
the  leucocyte  has  just  been  burst  open  and  the  streptococci  are  beginning 
to  grow  out  of  it;  in  Figure  3  a  considerable  colony  has  already  formed 
around  the  ruptured  leucocyte.  These  colonies  may  be  found  in  all  stages, 
by  varying  the  time  of  incubation. 


Fig.  2. — Photomicrograph  x^W-     Multiplication  of  streptococci  after  their 
ingestion  (in  vivo)  by  a  guinea-pig's  leucocyte. 


The  leucocytes  do,  however,  kill  streptococci  if  the  conditions  are 
right,  as  is  shown  by  the  following  two  facts:  i.  Organism  No.  300  is 
not  virulent  for  guinea-pigs,  and  we  have  seen  that  the  leucocytes  take' 
up  the  cocci  alive.  The  guinea-pig  must  destroy  these  organisms  some- 
where, and  we  can  find  no  substance  in  its  body  that  is  capable  of  doing 
this  except  the  leucocytes  and  the  bone  marrow.  We  have  seen  in  Table 
2  that  the  leucocytes  destroy  streptococci  in  vitro,  and  the  inference  is- 
that  they  also  destroy  them  in  vivo.  2.  Plates  made  with  three  loopfuls 
of  the  exudate  when  taken  from  the  guinea-pig  contained  2,540  colonies 
of  streptococci  after  twenty-four  hours'  incubation,  while  plates  made 


10 

with  three  loopfuls,  after  the  exudate  had  stood  in  a  test  tube  in  the 
incubator  for  six  hours,  contained  only  480  colonies.  Evidently  some  of 
the  streptococci  were  killed  in  this  tube,  but  the  smears  show  that  all  of 
the  organisms  are  in  the  leucocytes,  hence  they  must  have  been  killed 
in  the  leucocytes. 

EXPERIMENTS   WITH    LEUCOCYTIC  EXUDATE  IN   VIVO. 

In  order  to  determine  whether  a  rabbit  is  more  resistant  when  in- 
jected in  a  pleura  containing  leucocytic  exudate  than  when  injected  in  the 
normal  pleura,  the  following  experiments  were  carried  out : 


Fig.  3. — Photomicrograph  x  ]50°.     Multiplication  of  streptococci  after  their 
ingestion  (in  vivo)  by  a  guinea-pig's  leucocyte,  in  slightly  later  stage  than  Fig- 


ure 2. 


Experiment  A. — A  gray  rabbit  weighing  1,450  grams  was  injected 
into  the  right  pleura  with  7  c.  c.  of  aleuronat  suspension.  Fifteen  hours 
later  2,  c.  c.  of  a  4<>hour  culture  of  a  virulent  streptococcus  were  injected 
into  this  pleura  and  also  into  the  right  pleura  of  a  normal  rabbit  weigh- 
ing 1,500  grams.  The  normal  rabbit  was  found  dead  in  18  hours,  and 
the  one  which  had  been  previously  injected  with  aleuronat  died  45  hours 
later. 

Experiment  B. — A  gray  rabbit  weighing  2,100  grams  was  injected  in 
the  right  pleura  with  6  c.  c.  of  aleuronat  suspension.  Eighteen  hours 
later  i  c.  c.  of  a  24-hour  culture  of  a  virulent  streptococcus  was  injected 


11 

into  the  pleura  and  also  into  the  right  pleura  of  a  normal  gray  rabbit 
weighing  2,150  grams.  The  normal  rabbit  died  in  about  18  hours,  while 
the  aleuronat  rabbit  died  only  after  45  hours — 21  hours  after  the  death 
of  the  control. 

The  experiment  was  now  varied  so  that  the  conditions  were  the  same 
as  in  Simon's  experiments. 

Experiment  C. — A  rabbit  weighing  1,700  grams  was  injected  in  the 
right  pleura  with  8  c.  c.  of  aleuronat  suspension  and  48  hours  later  this 
rabbit  and  a  normal  rabbit  weighing  1,700  grams  were  each  injected  in 
the  right  pleura  with  I  c.  c.  of  a  3O-hour  streptococcus  culture.  In  this 
experiment  the  rabbit  treated  with  aleuronat  died  in  24  hours,  while  the 
control  lived  for  five  days. 

Experiment  D. — A  rabbit  weighing  1,500  grams  was  injected  in  the 
right  pleura  with  9  c.  c.  of  aleuronat  suspension.  Forty-eight  hours 
later  this  rabbit  and  a  normal  rabbit  weighing  1,800  grams  were  each 
injected  in  the  right  pleura  with  1.5  c.  c.  of  a  24-hour  streptococcus  cul- 
ture. The  normal  rabbit  died  in  37  hours,  while  the  rabbit  treated  with 
aleuronat  died  in  45  hours — eight  hours  after  the  death  of  the  control. 

The  results  of  the  last  two  experiments  agree  fairly  well  with  those 
obtained  by  Simon,  and  we  may  now  ask  why  they  are  different  from 
the  results  of  Experiments  A  and  B.  Apparently  this  difference  is  due 
to  the  fact  that  in  Experiments  C  and  D  the  inoculation  was  made  some 
thirty  hours  later  than  in  Experiments  A  and  B. 

That  this  is  the  correct  explanation  is  indicated  by  the  following  ex- 
periment : 

Experiment  E. — A  rabbit  weighing  2,300  grams  was  injected  in  the 
right  pleura  with  9  c.  c.  of  aleuronat  suspension.  Forty-eight  hours 
after  this  injection  the  rabbit  was  bled  to  death,  and  on  opening  the 
pleural  cavity  it  was  found  to  contain  no  leucocytic  exudate. 

Had  this  rabbit  been  inoculated  in  the  right  pleura  forty-eight  hours 
after  the  injection  of  the  aleuronat  suspension,  the  inoculation  would  not 
have  been  made  into  leucocytic  exudate.  That  being  the  case  we  would 
not  expect  that  this  rabbit  could  stand  a  larger  dose  of  streptococcus 
than  a  normal  rabbit.  In  Simon's  experiments,  however,  the  inoculations 
into  the  pleural  cavity  were  made  forty-eight  hours  after  the  injection 
of  aleuronat,  and  this  is  probably  the  reason  w'hy  he  did  not  find  these 
rabbits  more  resistant  than  normal  rabbits.  Had  he  made  his  inocula- 
tions within  twenty-four  hours  after  the  injection  of  aleuronat  his  re- 
sults would  very  probably  have  been  different*. 

At  the  suggestion  of  Professor  Hektoen  I  tested  filtrates  of  virulent 
streptococci  to  determine  whether  they  are  toxic  for  leucocytes,  as  has 


12 

been  shown  by  Van  de  Velde,*  and  by  Neisser  and  Wechsberg,**  to  be 
the  case  with  staphylococcus  filtrates.  The  organism  used  in  these  experi- 
ments was  BiO4,  which  has  been  passed  through  78  rabbits,  and  is  very 
virulent  for  rabbits  and  for  guinea-pigs,  and  produces  an  active  hemoly- 
sin  when  grown  in  heated  rabbit  serum  or  in  a  mixture  of  equal  parts 
of  rabbit  serum  and  ascites  fluid.  The  filtrates  used  were  from  iwenty- 
four  to  thirty-six-hour  cultures  in  a  mixture  of  rabbit  serum  and  ascites 
fluid,  which  has  been  heated  to  560.  for  one-half  hour  before  inoculation. 

Neisser  and  Wechsberg  have  shown  that  living  leucocytes  reduce 
methylene  blue  solution  when  the  supply  of  oxygen  is  shut  off,  while 
dead  leucocytes  no  longer  possess  this  property.  In  my  experiments  this 
fact  was  made  use  of  to  determine  whether  or  not  the  leucocytes  had 
been  killed  by  the  filtrate.  0.3  c.  c.  to  0.4  c.  c.  of  leucocytic  exudate  from 
a  rabbit  was  added  to  1.5  c.  c.  of  ~the  filtrate,  incubated  for  two  hours, 
then  two  drops  of  methylene  blue  solution  added  and  the  contents  of  the 
tube  shaken  up  and  covered  with  a  layer  of  sterile  olive  oil.  If  there 
was  reduction  in  two  hours  the  result  was  considered  negative,  but  if 
there  was  no  reduction  in  that  time  this  fact  was  taken  as  evidence  that 
the  leucocytes  were  dead.  Control  tubes  with  a  mixture  of  equal  parts 
of  heated  rabbit  serum  and  ascites  fluid  were  always  made,  and  the 
results  recorded  only  when  there  was  marked  reduction  in  the  control 
in  two  hours.  A  large  number  of  these  experiments  were  carried  out, 
but  it  is  not  necessary  to  tabulate  more  than  one  of  them  here. 

Experiment. — .3  c.- c.  exudate  +  1.5  c.  c.  24-hour  filtrate;  no  re- 
duction in  2  hours,  but  a  trace  of  reduction  in  3  hours. 

.3  c.  c.  exudate  -|-  1.5  c.  c.  36-hour  filtrate;  no  reduction  in  3  hours. 

.3  c.  c.  exudate  4-  1.5  c.  c  ascites-serum  ;*  nearly  complete  reduction 
in  2  hours. 

The  leucocytic  exudate  was  obtained  from  the  pleura  of  a  rabbit 
which  had  been  previously  injected  with  aleuronat  suspension  The 
methylene  blue  solution  was  made  according  to  the  formula  given  by 
Neisser  and  Wechsberg,  which  is  given  below : 

Solution  A. — Methylene  blue,  i;  absolute  alcohol,  20;  distilled 
water,  29. 

Solution  B. — i  c.  c.  of  solution  A ;  49  c.  c.  of  physiologic  salt  solu- 
tion. 

Two  drops  of  solution  B,  which  must  be  sterile,  are  added  to  each 
tube  in  the  experiments. 

*An.  de  1'Inst.  Pasteur,  389G,  X,  580. 
**Zcitschr.  f.  Hyg.,  1001.  XXX VI,  209. 

*The  term  "asdles-serum"  as  used  here  refers  to  a  mixture  of  equal  parts  of 
ascites  fluid  and  rabbit  serum,  heated  to  55  €.  for  one-half  hour. 


13 

The  fact  that  the  filtrates  have  a  decided  effect  on  leucocytes  has 
also  been  shown  in  a  second  experiment. 

Experiment. — Two  cubic  centimeters  of  a  rich  leucocyitc  exudate 
were  placed  in  each  of  five  small  test  tubes  and  centrifugated.  The  clear, 
supernatant'  fluid  was  now  decanted  from  each  tube  and  replaced  by  2 
c.  c.  of  bouillon,  heated  ascites-serum  or  filtrate  from  a  24-hour  ascites- 
serum  streptococcus  culture.  The  leucocytes  in  each  tube  were  evenly 
suspended  in  the  fluid,  the  tubes  placed  in  a  water  bath  at  37  C.  and  kept 
there  for  I  to  il/2  hours.  Now  .3  c.  c.  or  normal  rabbit  serum  (guinea- 
pig  serum  when  guinea-pig's  leucocytes  were  used)  was  added  to  each 
tube,  the  tubes  inoculated  with  a  loopful  of  a  non-virulent  streptococcus 
broth  culture  and  plates  made  at  intervals,  the  tubes  being  kept  in  the 
incubator. 

The  following  table  shows  the  results  of  an  experiment: 

Immed.  4-5  hrs.  20  hrs. 

Leucocytes  -f  2  c.  c.  filtrate  B104  +  .3  c.  c.  normal  serum.  .2000      3200  Many 

Leucocytes  -f  2  c.  e.  filtrate  10  A  +  .3  c.  c.  normal  serum  .  .1200      2000  Many 

Leucocytes  +  2  c.  c.  filtrate  300  +  .3  c.  c.  normal  serum  ..1100          24  Many 

Leucocytes  +  2  c.  c.  ascites  serum  -j-  .3  c.  c.  normal  serum  .  .1000         140  Many 

Leucocytes  -f-  2  c.  c.  bouillon  -f-  .3  c.  c.  normal  serum 1800          GO  Many 

The  numbers  B10-1,  10A  and  300  refer  to  different  strains  of  streptococci. 
B104  has  a  high  virulence  and  10A  a  medium  virulence;  both  these  strains  pro- 
duce liemolysin  in  heated  serum,  although  10A  has  never  been  passed  through 
animals.  It  was  recently  isolated  from  suppurating  axillary  glands.  300  is 
non-virulent,  and  does  not  produce  hemolysin. 

The  suspensions  of  leucocytes  in  filtrates  from  serum  cultures  of 
virulent  streptococci  have  no  effect  on  streptococci  while  the  suspension 
in  filtrates  from  non-virulent  organisms,  in  heated  ascites-serum,  and  in 
bouillon  destroy  large  numbers  of  cocci. 

These  experiments  do  not  always  give  positive  results,  but  there  is 
a  small  percentage  of  tubes  in  which  the  filtrate  has  apparently  no  effect 
on  the  leucocytes.  This  is  not  surprising,  however,  when  we  consider 
the  fact  that  in  about  .TOO  filtrates  which  I  have  tested  for  hemolysis, 
there  were  nearly  10  per  cent,  that  had  no  hemolytic  properties,  whereas 
some  of  them  were  so  active  that  .005  c.  c.  completely  laked  i  c.  c.  of  a 
2]/2  per  cent,  suspension  of  rabbit's  washed  corpuscles  in  two  hours. 

TEST  TUBE  EXPERIMENTS  WITH  HUMAN  LEUCOCYTES. 

The  foregoing  experiments  show  that  the  leucocytes  and  opsonin  are 
the  most  important,  if  not  the  only,  factors  concerned  in  the  destruction  of 
streptococci  in  the  body  of  infected  rabbits  and  guinea-pigs.  In  strepto- 


14 

coccus  infections  in  man  the  tissues  are  invaded  by  virulent  streptococci 
but  in  a  majority  of  these  cases  the  cocci  disappear  sooner  or  later  and  the 
patients  make  a  complete  recovery.  Freshly  drawn  human  serum  has  no 
streptococcidal  powers  in  vitro  and  it  is  reasonable  to  suppose  that  it  has 
none  in  the  body  during  life.  We  must  look  therefore  for  some  other 
agent  than  the  serum  alone  to  account  for  the  disappearance  of  the  cocci 
from  the  tissues  during  convalescence.  According  to  Metchnikoff  and 
his  followers  this  agent  is  found  in  the  phagocytes  and  this  view  seems  to 
explain  the  facts  better  than  any  other  theory  that  has  been  advanced. 

I  next  undertook  a  series  of  experiments  to  determine  more  accu- 
rately what  factors  are  concerned  in  the  destruction  of  the  invading 
cocci  in  cases  of  human  streptococcus  infections,  and  to  analyze  more 
fully  this  phenomenon.  In  all  instances  where  blood  was  used  it  was 
drawn  from  the  vein  at  the  elbow  by  means  of  a  Luer  syringe  and  de- 
fibrinated  by  gently  whipping  with  a  sterile  wire.  If  the  defibrinating 
is  carefully  performed  not  a  very  large  proportion  of  the  leucocytes  are 
destroyed.  In  the  experiments  each  tube  contained  from  0.8  to  i.o  c.  c. 
of  blood  or  serum  which  was  inoculated  with  one  loopful  of  streptococcus 
culture  and  two  loopfuls  from  each  tube  were  plated  in  glucose  agar  at 
intervals.  The  tubes  were  always  kept  in  the  incubator  at  36  C.  When 
highly  virulent  organisms  were  used  0.3  to  0.4  c.  c.  of  defibrinated  rabbit 


TABLE  6. 

THE  EFFECT  OF   DEF1B1IINATED   NORMAL   HUMAN   HLOOD  AND   HUMAN    SERUM    ON    STREP- 
TOCOCCI. 


Strepto- 
cocci. 

300 

300 

300 

300 

300 

300 

300 

298 

298 

298 
B104 
B104 
B104 
B104 

381 

300 

298 


Defibrinated 
Blood. 
I 

II 

I  la 

III 

IV 

V 

VI 

VII 

VII 

VIII 

I  la 

III 

IV 

V 

VI 

Serum  IV 
Semin  VII 


Colonies 

in  Agar  Plates. 

limned.    2 

to  3  hours. 

1100 

.... 

3000 

.... 

680 

675 

2600 

2700 

3000 

1050 

540 

360 

1100 

500 

1600 

600 

1600 

600 

600 

80 

76 

56 

1800 

2800 

1600 

3000 

500 

600 

2000 

2300 

1400 

2000 

1700 

2500 

5  hours. 

160 

1060 

1300 

3500 

2600 

240 

390 

315 

315 

16 

510 

Many 

Many 

900 

Many 

Many 

10000 


15 

blood  was  added  to  each  tube  of  melted  agar  to  facilitate  the  counting 
of  colonies,  which  often  are  very  small  if  no  blood  has  been  added. 

Table  6  shows  that,  although  human  serum  in  vitro  is  a  good  culture 
medium  for  streptococci,  normal  defibrinated  blood  has  a  slight  strepto- 
coccidal  power.  Occasionally  we  may  find  a  sample  of  normal  blood 
which  destroys  many  non-virulent  streptococci,  but  the  virulent  organ- 
isms usually  multiply  in  this  blood.  Table  /  shows  that  defibrinated 
blood  from  patients  suffering  from  an  acute  infection  has  a  much  greater 
destructive  effect  on  these  organisms  than  has  normal  blood.  (The  non- 
virulent  cocci  are  ingested  very  freely  by  the  leucocytes  while  the  viru- 
lent strain  is  not  taken  up  so  freely,  as  shown  by  counts  in  stained 
smears.) 


TABLE   7. 

THE    EFFECT    OF    DEFIBRIX.VTKH    PLOOD    FROM    CASES    OF    SCARLATINA,    ERYSIPELAS    AND 


PNEUMONIA   ON    STREPTOCOCCI. 


Strepto- 
cocci. 

300 

?>00 

300 

300 

300 

300 

300 

300 

300 

300 

300 

300 

300 

300 

381 
B104 
B104 

381 

381 

381 

381 
B104 

381 
B104 

381 

381 

381 


Colonies 


Defibri  nated  Leucocyte 

Blood.  count.           Immed. 

Scarlatina              I  10500  1100 

II  10600  2000 

III  15000  690 

IV                 550 

V                 1500 

VI                  1000 

VII                  500 

VIII  13000  1380 

IV                  1200 

X  13400  1250 

XI                  1950 

XII  10800  1800 

XIII                  1800 

XIV  12300  1500 

III  15000  1200 

IV                 600 

V                 1800 

VI                 220 

VII                  130 

VIII  13000  360 

IX  13000  2200 

IX                  330 

X  13400  *640 

X                  400 

X                 320 

XII  10800  200 

XIII  540 


in  Agar 
2  to  3 
hours. 


270 

18 
300 
350 
32 
102 
125 
245 
150 
700 
300 
31 

1500 

462 

1800 

51 

41 

1180 

1950 

190 

290 

160 

650 

2000 

900 


Plates. 

5  hours. 
6 

140 

116 

3 

150 

43 

12 

9 

14 

120 

19 

130 

45 

2 

3500 

415 

5000 

1100 

Many 

Many 

Many 

1500 

2500 

165 

Many 

Many 

Many 


16 

300  Erysipelas      I  .....  G30  64  0 

300  II  15000  800  14  4 

300  H;i  1100  41  8 

300  III  740  120  160 

300  IV  1650  480  150 

298  V  10400  3100  420  02 

I  1200  1080  960 

B104  II  96  30  13 

B104  IV  133  110  480 

300  Tonsillitis                 I  16000  620  34  2 

JB104  I  300  59  70 

300  II  1400  74  6 

300  Pneumonia             1  2600  0  3 

500  "                     II  600  5  0 

Serum 

300  Scarlatina          VII  1500  3500  10000 

300  "                 XIV  1200  1400  1300 

300  Erysipelas            II  670  760  7.".0 

300  III  700  1800  6<XIO 

300  "               .V  3000  3500  5000 

B104  "                       V  190  360  1140 

In  these  infections  the  leucocyte  count  is  usually  somewhat  increased, 
and  it  seems  that  the  streptococcidal  power  of  the  blood  in  vitro  is 
roughly  proportional  to  the  leucocyte  count.  That  is,  the  higher  the  leu- 
cocyte count  the  greater  will  be  the  streptococcidal  power  of  the  blood. 
This  is  a  general  rule;  to  which  there  are,  however,  a  few  exceptions, 
as  will  be  pointed  out  later.  The  virulent  organisms  frequently  multiply 
in  these  bloods  unless  the  leucocytosis  is  very  high.  In  no  instance  could 
a  streptococcidal  power  of  the  serum  alone  be  detected.  It  might  be 
objected  that  we  are  not  dealing  here  with  an  actual  destruction  of  cocci, 
but  that  the  decrease  in  the  number  of  colonies  on  the  plates  is  due  to 
adherence  of  the  cocci  to  the  leucocytes.  This  objection  is  ruled  out 
by  the  fact  that  the  twenty-four-hour  plates  from  tubes  containing  blood 
with  a  high  leucocytosis  are  very  often  sterile  or  nearly  so. 

It  has  been  thought  possible  that  the  serum  during  the  course  of  an 
infection  which  terminates  favorably  might  acquire  streptococcidal  prop- 
-erties  for  that  particular  race  of  streptococci  which  is  responsible  for 
•the  infection,  while  at  the,  same  time  it  had  no  such  properties  for  other 
races  of  these  organisms.  Three  strains  of  streptococcus  were,  there- 
fore, isolated  from  erysipelas  patients  and  the  serum  of  each  patient 
tested  on  the  corresponding  organism.  All  of  the  patients  made  a  sat- 
isfactory recovery,  but  at  no  time  could  streptococcidai  properties  be 
demonstrated  in  their  serum.  The  defibrinated  blood,  on  the  other  hand, 


17 

killed  many  of  the  homologous  and  other  organisms  as  long  as  there 
was  a  high  leucocytosis. 

We  know  that  there  is  an  intense  local  reaction  in  the  localized 
streptococcus  infections,  and  it  has  been  thought  by  some  that  there  may 
be  lysis  of  cocci  by  the  inflammatory  serum  in  these  areas.  It  is  difficult 
to  confirm  or  refute  this  theory  on  account  of  the  difficulty  of  obtaining 
inflammatory  serum  in  the  same  condition  as  it  is  found  in  the  tissues. 
As  it  is  not  uncommon  to  find  blebs  of  considerable  size  on  the  affected 
parts  of  erysipelas  patients,  the  fluid  from  these  blebs  was  taken  as  the 
nearest  approximation  to  the  inflammatory  serum.  This  blister  fluid 
from  several  cases  of  erysipelas  was  tested  for  streptococcidal  proper- 
ties but  gave  negative  results. 

The  importance  of  a  high  leucocyte  count  in  the  destruction  of 
streptococci  by  blood  is  clearly  shown  by  the  following  experiment: 

Experiment  i.  Ten  cubic  centimeters  of  blood  were  drawn  from 
the  vein  at  the  elbow  of  an  erysipelas  patient  and  carefully  defibrinated. 
One  cubic  centimeter  of  the  defibrinated  blood,  which  contained  9,800 
white  corpuscles  per  cubic  millimeter,  was  put  into  a  small  test  tube, 
inoculated  with  one  loop f til  of  virulent  streptococcus  culture,  and  two 
loopfuls  of  the  inoculated  blood  were  plated  at  intervals.  The  remaining 
eight  cubic  centimeters  were  centrifugated  and  the  serum  drawn  off. 
We  know  that  the  uppermost  stratum  of  centrifugated  corpuscles  con- 
tains a  high  percentage  of  leucocytes,  because  they  are  thrown  down  less 
easily  than  the  red  corpuscles.  This  stratum  was  therefore  drawn  off 
with  a  sterile  pipette  and  mixed  with  a  small  quantity  of  serum.  The 
resultant  mixture  contained  17,200  leucocytes  per  cubic  millimeter.  One 
cubic  centimeter  of  this  "suspension  of  leucocytes"  was  introduced  into 
a  small  test  tube,  inoculated  and  plates  made  as  before.  To  complete 
the  experiment  one  cubic  centimeter  of  the  clear  serum  was  put  into  a 
small  tube,  which  was  likewise  inoculated,  and  plates  made  at  intervals. 
This  experiment  was  also  performed  with  normal  blood  and  a  non-viru- 
lent streptococcus.  The  plates  were  incubated  for  twenty-four  hours, 

TABLE    8. 

Colonies  on  Agar  Plates. 

Strepto-  Leucocyte 

cocci.  count.  Immed.  2  to  3  lirs.  5  hrs. 

B104         Erysipelas  Wood   9800          390  108  350 

B104         Suspension  erysipelas  leuc 17200          260  58  21 

B104        Erysipelas  serum * 200  360  1100 

298         Normal  blood 4400         1650  600  315 

298         Suspension  normal  leuc 6600        1600  420  62 

298         Normal  serum   .                                                                1700  2500  10000 


IS 

and  the  colonies  that  developed  on  each  were  counted  with  the  results 
shown  in  Table  8. 

The  table  shows  that  both  strains  of  streptococcus  used  multiplied 
in  the  cell- free  serum;  that  the  defibrinated  blood  destroyed  many  of 
the  non-virulent  and  some  of  the  virulent  cocci,  and  the  "suspension  of 
leucocytes"  destroyed  more  cocci  of  either  strain  than  the  defibrinated 
blood.  The  only  difference  between  the  defibrinated  blood  and  the  "sus- 
pension of  the  leucocytes"  lay  in  the  fact  that  the  latter  contained  nearly 
twice  as  many  leucocytes  as  the  former. 

The  fact  that  the  streptococcidal  power  of  the  blood  is  dependent 
on  the  number  of  leucocytes  it  contains  per  cubic  millimeter  has  also 
been  demonstrated  by  a  second  experiment. 

Experiment  2. — Shortly  after  a  patient's  admission  into  the  hospital, 
3  c.  c.  of  blood  was  drawn  from  a  vein  at  the  elbow  and  defibrinated. 
The  leucocyte  count  at  this  time  was  11,000.  The  blood  was  divided 
equally  among  three  tubes,  and  each  tube  was  inoculated  with  strepto- 
coccus culture  and  two  loopfuls  from  each  were  plated  at  intervals. 
Shortly  after  drawing  the  blood,  the  patient  was  injected  under  the  skin 
of  the  back  with  TO  c.  c.  of  an  antistreptococcus  serum.  This  brought 
about  an  increase  in  the  leucocytes  up  to  15,000,  five  hours  after  the  in- 
jection. Three  c.  c.  of  blood  was  again  drawn  from  the  vein  at  the  elbow 
and  its  effect  on  streptococci  tested  as  before.  The  results  of  the  experi- 
ments are  shown  in  Table  9. 

TABLE   9. 

Colonies  on  Agar  Plates, 

Strepto-  Leucocyte 

cocci.  count.  Immecl.  2  to  3  hrs.  5  hrs. 

800        Blood  before  injection 11000        1100  360            200 

B104        Blood  before  injection   390  380          4000 

381        Blood  before  injection    , 100  500        Many 

300        Blood  5  hours  after  injection  15000        1300  44                0 

B104        Blood  5  hours  after  injection   412  240            600 

381        Blood  5  hours  after  injection   150  270          3000 

The  blood  drawn  after  the  injection  of  the  serum,  when  the  leucocy- 
tosis  was  high,  has  a  greater  streptococcidal  power  than  that  drawn  before 
the  injection.  It  would  not  be  safe  to  conclude  that  this  difference  in  the 
streptococcidal  power  is  due  entirely  to  the  difference  of  the  leucocyte 
count.  Some  of  it  may  be  due  to  an  antitoxic  or  opsonic  action  of  the 
serum  or  to  a  stimulation  of  the  leucocytes.  This  supposition  loses  most 
of  its  force  when  we  consider  the  fact  that  the  addition  of  from  I  to  5 
per  cent  of  this  antistreptococcus  serum  to  defibrinated  blood  in  vitro 


19 

does  not  increase  its  streptococcidal  power,  as  shown  by  experiments  by 
Hektoen  &  Ruediger. 

We  know  that  untreated  bacteria  are  not  taken  up  by  washed  leu- 
cocytes in  Na  Cl  solution  or  in  heated  serum,  and  hence  there  should  be 
no  reduction  in  the  number  of  streptococci  in  a  test  tube  containing  a 
suspension  of  washed  human  corpuscles  in  salt  solution  or  in  heated 
serum.  The  following  experiment  shows  that  this  is  the  case : 

Experiment  3. — Ten  c.  c.  of  blood  was  drawn  from  a  vein  at  the 
elbow  of  a  scarlet  fever  patient,  defibrinated,  centrifugated  and  the 
serum  drawn  off.  The  corpuscles  were  washed  twice  in  a  large  amount 
of  NaCl  solution,  and  0.5  c.  c.  of  the  washed  corpuscles  placed  into 
each  of  three  small  tubes  containing  0.5  c.  c.  of  normal  serum,  0.5 
c.  c.  of  heated  serum  (58  degrees  for  one-half  hour)  and  0.5  c.  c.  of 
salt  solution,  respectively.  The  tubes  were  inoculated  with  one  loopful 
of  streptococcus  culture,  and  two  loopfuls  from  each  were  plated  at  in- 
tervals with  the  results  shown  in  Table  10. 

TABLE   10. 

Colonies  on  Glucose  Agar  Plater.. 
Strepto- 
cocci.                                                                                           Immed.  5  hours.  24  hours.. 

300        Washed  corpuscles  +  serum 1500                  7  21 

300        Washed  corpuscles  +  heated  serum 1400            5000  Many 

300        Washed  corpuscles  +  NaCl  solution  1700            3500  Many 

300         Serum   1200            1400  Many 

The  importance  of  opsonin  in  the  destruction  of  streptococci  by  hu- 
man leucocytes  is  further  shown  by  the  fact  that  the  defibrinated  blood 
from  two  obstinate  cases  of  post-scarlatinal  nephritis  had  no  streptococci- 
dal powers,  although  in  one  of  the  cases  the  leucocyte  count  was  14,000. 
When  the  blood  from  these  patients  was  centrifugated  and  the  corpuscles 
suspended  in  serum  from  a  patient  who  was  convalescent  and  had  no 
nephritis,  the  resultant  suspension  had  a  small  degree  of  streptococcidal 
power,  as  shown  by  Table  n. 

The  interesting  fact  came  to  light  in  these  experiments  that  the 
combination  "convalescent  washed  corpuscles  plus  nephritis  serum''  has 

TABLE   11. 

Colonies  on  Agar  Plates. 
Strepto- 
cocci. Immed.  2  hours.  5  hours.. 

298        Convalescent  blood 800  450  14 

298        Nephritis  blood  720  1900  6000 

298        Nephritis  corpuscles  -j-  cony,  ser 850  500  540> 

298        Conv.  washed  corp.  -f  Neph.  ser 725  GOO  01 


20 

nearly  as  great  a  streptococcidal  power  as  the  defibrinated  blood  from 
the  patients  without  nephritis.  The  corpuscles  were  washed  twice  in  a 
large  amount  of  NaCl  solution  which  is  usually  sufficient  to  prevent 
phagocytosis  in  a  suspension  of  corpuscles  in  NaCl  solution.  It  is  not 
likely,  therefore,  that  the  washing  had  not  been  carried  far  enough.  But 
the  results  of  these  experiments  seem  to  indicate  rather  that  the  leuco- 
cytes as  well  as  the  serum  from  these  nephritis  patients  have  undergone 
some  change  which  renders  them  less  efficient  in  the  destruction  of  bac- 
teria. In  fact,  it  would  seem  that  the  leucocytes  have  suffered  more  than 
the  serum.  Whether  or  not  these  facts  may  serve  to  throw  light  on  the 
cause  of  some  of  the  terminal  infections  can  not  be  determined  at  this 
time. 

It  is  an  interesting  question  whether  the  opsonin  is  increased  or  not 
during  the  acute  infections.  Normal  leucocytes  in  normal  serum  take  up 
large  numbers  of  cocci ;  hence,  it  is  difficult  to  determine  if  leucocytes  in 
erysipelas  serum,  for  instance,  take  up  more  cocci  than  those  in  normal 
serum.  This  question  had,  therefore,  to  be  approached  in  a  different 
way. 

Experiment  4. — Two  sets  of  tubes  were  made  and  0.2  c.  c.  of  washed 
corpuscles  introduced  into  each.  To  one  set  of  tubes  were  added  falling 
quantities  of  normal  serum  and  to  the  other  set  falling  quantities  of 
•erysipelas  serum.  The  contents  of  each  tube  were  made  up  to  0.4  c.  c. 
with  NaCl  solution,  and  to  each  tube  was  added  0.4  c.  c.  of  a  suspension 
of  streptococci.  The  tubes  were  incubated  for  one  hour  at  36  C.,  smears 
were  made  and  the  average  number  of  cocci  in  each  leucocyte  determined 
"by  counting  those  in  30  leucocytes.  The  results  are  shown  in  Table  12. 

Table   12. 

Normal  serum.              Phagocytosis.  Erysipelas   serum.              Phagocytosis. 

0.2       c.  c 13.5  0.2       c.  c 11.5 

0.1       c.  c 0.4  0.1       c.  c 11.2 

0.05     c.  c 0.7  0.05     c.  c 8.2 

0.025  c.  c 4.7  0.025  c.  c 0.0 

•0.012  c.  c 2.2  0.012  c.  c 4.3 

This  experiment  indicates  that  there  is  a  slight  increase  of  opsonin 
in  the  erysipelas  serum  as  compared  with  normal  serum.  A  similar  in- 
crease of  opsonin  has  been  noted  by  Wright  and  Douglas*  after  treat- 
ing with  their  staphylococcus  vaccine  a  person  afflicted  with  furunculosis. 
Hektoen**  also  observed  a  rise  in  opsonin  after  injecting  a  person  with 

**Proc.  Royal  Sue.,  1904. 

**Journ.  A.  M.  A.,  1006,  XLVI,  1400. 


21 

heated  streptococci.  A  diminution  of  opsonin  in  persons  subject  to  at- 
tacks of  furunculosis,  sycosis,  etc.,  has  also  been  observed  by  Wright  and 
Dong-las. 

During  the  course  of  this  work  I  had  an  opportunity  to  study  a 
case  of  acute  purulent  rhinitis,  in  the  discharge  of  which  streptococci 
were  found  in  pure  culture. 

Patient. — A  man  27  years  of  age.  During  the  first  two  days  the 
discharge  was  clear  and  watery  and  contained  very  many  streptococci  in 
short  chains  and  in  diplococci.  Figure  4  shows  a  photomicrograph  of  a 
smear  make  on  the  second  day.  There  were  only  a  few  leucocytes,  and 
those  did  not  contain  any  streptococci.  On  the  fourth  day  the  discharge 


Fig.  4. — Photomicrograph  X   1500.     Streptococci  hi  nasal  discharge  on  sec- 
ond day  of  sickness. 


had  become  thick  and  purulent,  and  smears  made  at  that  time  presented 
an  entirely  different  picture.  There  were  no  free  streptococci,  but  very 
many  leucocytes,  some  of  which  were  loaded  with  streptococci.  Figure 
5  shows  a  photomicrograph  of  such  a  preparation  made  on  the  fourth 
day.  The  purulent  discharge  continued  for  about  a  week,  and  during 
this  time  it  was  easy  to  find  leucocytes  in  the  smears  which  contained 
large  numbers  of  streptococci.  The  streptococci  finally  disappeared,  and 
the  patient  made  a  complete  recovery. 

In  this  case  of  streptococcus  infection  phagocytosis  of  the  organ- 
isms by  the  leucocytes  seemed  to  be  an  important  factor  in  combating 


22 

the  infection.  In  the  smears  made  on  the  fourth  day  no  free  streptococci 
could  be  found,  although  diligent  search  was  made  for  them.  Plates 
made  with  a  small  quantity  of  discharge,  which  was  obtained  high  up  in 
the  nares  by  means  of  a  sterile  cotton  swab,  contained,  however,  many 
colonies  of  streptococci  after  twenty- four  hours'  incubation.  The  fact 
that  many  colonies  developed  in  the  plates,  although  no  free  streptococci 
could  be  found  in  the  smears,  seems  to  indicate  that  the  organisms  were 
not  dead  when  they  were  taken  up  by  the  phagocytes,  but  were  taken 
tip  alive.  It  is  possible,  however,  that  there  were  free  streptococci  in  this 
purulent  discharge,  even  though  they  were  not  found  in  the  smears. 


Fig.  5. — Photomicrograph  X   1500.     Streptococci  in  a  leucocyte  from  nasal 
discharge  on  fourth  day  of  sickness. 


IMMUNIZATION  OF  RABBITS  WITH  STREPTOCOCCI. 

There  can  no  longer  be  any  doubt  about  the  statements  of  many  in- 
vestigators that  laboratory  animals  acquire  a  fair  degree  of  immunity 
after  repeated  injections  of  small  doses  of  virulent  streptococci.  I  have 
injected  a  large  number  of  rabbits  first  with  several  doses  of  heated  cul- 
tures (65°  C.  for  20  minutes)  and  then  with  living  cultures  of  medium 
virulence,  and  have  found  that  some  animals  acquire  enough  resistance 
after  4  or  5  injections  to  withstand  injections  of  several  times  the  mini- 
mum fatal  dose  of  the  streptococcus.  Thus,  for  instance,  a  full  grown 
rabbit  weighing  2000  G.  had  received  2  large  injections  of  heated  culture 


23 

and  4  small  injections  of  the  living  culture  "381"  at  intervals  of  8  to  10 
days.  This  rabbit  was  now  injected  under  the  skin  of  the  back  with  4 
c.  c.  of  a  suspension  of  streptococcus  "381"  and  at  the  same  time  a  nor- 
mal rabbit  weighing  2100  G.  was  injected  in  the  corresponding  locality 
with  2  c.  c.  of  the  same  streptococcus  suspension.  The  normal  rabbit 
died  on  the  3rd  day  while  the  immune  rabbit  showed  no  signs  of  sickness 
except  that  it  lost  150  G.  in  weight,  which  was  regained  at  the  end  of 
one  week. 

Experiments  with  rabbit  leucocytes  in  immune  serum  in  vitro.  Ex- 
periments were  now  undertaken  to  make  a  further  study  of  the  mechan- 
ism of  this  immunity  than  has  already  been  made  by.Neufeld  and  Rimpau 
and  others. 

An  immune  rabbit  and  a  normal  rabbit  were  each  bled  5  c.  c.,  the 
bloods  defibrinated,  divided  into  two  equal  quantities  and  leucocytes 
added  to  each  blood.  One  portion  of  each  blood  received  leucocytes 
from  the  normal  rabbit  and  the  other  portion  received  washed  leucocytes 
from  the  immune  rabbit.  The  whole  blood  instead  of  the  serum  alone 
was  used  because  rabbit  blood  contains  only  a  very  small  number  of 
polymorphonuclear  leucocytes  which  may  be  neglected,  and  it  has  been 
found  that  smears  from  tubes  containing  leucocytes  in  blood  are  more 

TABLE  13. 

PHAGOCYTOSIS   OF    STREPTOCOCCI  BY   NORMAL  AND  BY  IMMUNE  RABBIT   LEUCOCYTES    JN 
NORMAL  AND  IN  IMMUNE  RABBIT  BLOOD. 

Strepto-  Phagocytosis  by  normal  leucocytes  in : 

cocci.                                             Normal  Blood.  Immune  Blood. 

Laura*  G.2  14. 

381  2.2  4.8 

Joe  R.  4.4  7.G 

Stuart  1.  4.0 

Puerperal  1.  1.6 

Phagocytosis  by  normal  leucocytes  in  : 

Normal  Blood.  Immune  Blood. 

Laura                                                        5.5  15.2 

381                                                             1.7  5. 

Joe  R.                                                        5.3  7.2 

Stuart                                                        1.  4.2 

Puerperal                                                 5.  2.5 


**Sources  of  Streptococci:  "Laura"  from  a*  scarlatinal  otitis  media,  and 
used  to  immunize  the  rabbit,  the  serum  of  which  was  used  hi  this  experiment; 
"Joe  R."  from  a  scarlatinal  sore  throat ;  "381"  from  pericardial  fluid  of  a  scarla- 
tinal body ;  "Stuart"  from  a  case  of  erysipelas  and  "Puerperal"  from  a  blood  cul- 
ture of  a  case  of  puerperal  sepsis.  All  were  only  of  medium  virulence. 


24 

satisfactory  than  those  made  from  suspensions  of  leucocytes  in  serum. 
Four-tenths  of  a  cubic  centimeter  of  each  suspension  of  leucocytes  in 
blood  was  put  into  a  number  of  small  test  tubes  arid  .2  c.  c.  of  a  fairly 
thick  suspension  of  virulent  streptococci  added  to  each  tube.  The  tubes 
were  incubated  at  36  C.  for  i  hour,  smears  were  made  and  the  average 
number  of  cocci  ingested  by  each  leucocyte  was  determined  by  counting 
the  cocci  in  30  leucocytes.  The  results  of  such  an  experiment  are  shown 
in  Table  13. 

The  Table  shows  that  there  is  a  larger  number  of  cocci  taken  up 
by  the  leucocytes  in  immune  blood  than  by  those  in  normal  blood;  that 
the  immune  leucocyfes  do  not  ingest  appreciably  more  cocci  than  the 
normal  leucocytes  and  that  the  increase  is  most  marked  with  that  strain 
of  streptococcus  which  was  used  in  immunizing  the  rabbit. 

Four  other  experiments  with  the  bloods  of  different  immune  rabbits 
gave  practically  the  same  results.  The  increased  phagocytosis  always 
was  most  pronounced  with  that  strain  of  streptococcus  which  had  been 
used  in  immunizing  the  rabbit  whose  defibrinated  blood  or  serum  was 
used  in  the  experiment.  Several  strains  of  streptococcus  were  used  in 
the  immunization  and  all  that  were  of  medium  virulence  gave  an  im- 
munity that  could  be  demonstrated  in  the  test  tube.  Rabbits  were  also 
treated  with  a  highly  virulent  streptococcus,  but  this  organism  was 
scarcely  taken  up  at  all  by  rabbit  leucocytes  suspended  in  this  or  in  other 
immune  sera. 

An  experiment  was  now  carried  out  to  show  how  the  immune  serum 
promoted  phagocytosis  of  the  virulent  cocci.  For  this  purpose  normal 
rabbit  leucocytes  were  suspended  in  normal  and  some  in  immune  rabbit 
serum  for  */?,  hour,  then  centrifugated  out  of  the  sera,  washed  twice  in 
NaCl  solution  and  suspended  in  normal  serum.  To  each  tube  was  now 
added  the  same  amount  of  suspension  of  a  virulent  streptococcus,  the 
tubes  were  incubated  for  i  hour,  smears  were  made  from  each  and  the 
degree  of  phagocytosis  determined  by  counts.  In  neither  tube  was  there 
any  phagocytosis,  showing  that  the  immune  serum  is  not  capable  of 
changing  the  leucocytes  so  that  they  will  ingest  virulent  streptococci 
which  have  not  been  treated  with  immune  serum.  A  second  pair  of 
tubes  was  carried  through  at  the  same  time,  using  the  same  leucocytes, 
streptococcus  suspension  and  sera.  In  this  instance  virulent  streptococci 
were  treated  J/£  hour  with  immune  rabbit  serum  and  some  with  normal 
rabbit  serum,  then  washed  once  in  a  large  amount  of  NaCl  solution,  sus- 
pended in  NaCl  solution  and  each  lot  added  separately  to  a  suspension 
of  normal  rabbit  leucocytes  in  NaCl  solution.  The  tubes  were  in- 
cubated i  hour,  smears  were  made  as  above  and  the  degree  of  phagocy- 
tosis determined  by  counts.  In  this  pair  of  tubes  the  degree  of  phago- 


25 

cytosis  was  strikingly  different.  The  cocci  which  had  been  sensitized  in 
normal  serum  were  not  ingested  by  the  leucocytes,  while  those  that  had 
been  sensitized  in  immune  serum  were  taken  up  freely.  That  is,  the 
serum  has  acquired  something  in  the  process  of  immunization  by  virtue  of 
which  it  is  capable  of  so  changing  the  virulent  streptococci  that  they 
become  susceptible  to  phagocytosis.  The  results  of  the  count  are  shown 
in  Table  14. 

TABLE   14. 

PHAGOCYTOSIS  OF  VIRULENT  STREPTOCOCCI  SENSITIZED  IN  IMMUNE  RABBIT   SERUM. 

1.  Washed  rabbit  leucocytes  +  normal  rabbit  serum  +  streptococcus 

"Laura"   0.4 

2.  Rabbit    leucocytes    suspended    in   immune    rabbit    serum    y2    hour, 

washed   in   NaCl   solution,   resuspended   in   normal   serum    + 
streptococcus    "Laura"    0.0 

3.  Washed  rabbit  leucocytes  in  NaCl  solution  +  streptococcus  "Laura" 

sensitized  in  normal   rabbit  serum    0.6 

4.  Washed  rabbit  leucocytes  in  NaCl  solution  +  streptococcus  "Laura" 

sensitized  in  immune  rabbit  serum   8.0 

The  destruction  of  virulent  streptococci  by  rabbit  leucocytes  in  im- 
mune serum  can  also  be  shown  by  means  of  plate  cultures.  For  this 
purpose  2  tubes  were  prepared  containing  (a)  I  c.  c.  defibrinated  nor- 
mal blood  +  .2  c.  c.  of  a  thick  suspension  of  rabbit  leucocytes,  and  (b) 
i  c.  c.  defibrinated  immune  blood  -(-  .2  c.  c.  of  the  same  suspension  of 
leucocytes.  Each  tube  was  inoculated  with  one  loopful  of  a  virulent 
streptococcus  culture,  incubated  at  36°  C.  and  plates  made  at  intervals 
with  2  loopfuls  of  blood  from  each  tube.  The  plates  were  incubated  for 
24  hours  and  the  colonies  carefully  estimated  with  the  following  result: 

Strepto-                                                                                      Colonies  in  Agar  plates, 

cocci.                                                                                           Immeil.  2  hours.  5  hours. 

381         Leucocytes  in  normal  blood  480  1200            SOOO 

381         Leucocytes  in  immune  blood 460  300  5000 

In  the  tube  containing  immune  blood  +  leucocytes  the  number  of 
•organisms  decreased  during  the  first  two  hours  but  then  began  to  in- 
crease rapidly,  while  in  the  other  tube  they  increased  rapidly  from  the 
beginning  of  incubation.  The  immune  serum  alone  has  no  destructive 
action  on  these  organisms  but  acts  as  a  culture  medium  for  them. 

The  following  experiment  was  now  made  to  determine  whether  the 
opsonin  of  the  immune  serum  can  withstand  a  higher  degree  of  heat 
than  that  of  normal  serum. 

Experiment. — .2  c.  c.  of  normal  serum  and  .2  c.  c.  of  immune  serum 
in  two  small  test  tubes  were  heated  to  60°  C.  for  y2  hour.  To  each 


26 

tube  were  now  added  .2  c.  c.  suspension  of  washed  rabbit  corpuscles 
containing  leucocytes  from  a  pleural  exudate  and  .2  c.  c.  suspension  of 
streptococci  of  medium  virulence.  Two  additional  tubes  with  unheatecl 
normal  and  immune  serum  were  prepared  in  the  same  manner  and  all 
tubes  incubated  ]/2  hour.  Smears  were  now  made  from  all  tubes  and 
stained  and  the  degree  of  phagocytosis  in  each  tube  determined  by  count- 
ing the  cocci  in  each  of  50  leucocytes,  with  the  following  result : 

Phagocytosis. 

Heated  normal  serum  ~j-   washed  corpuscles  +  streptococci   . ! 1.1 

Heated  immune  serum  +  washed  corpuscles  +  streptococci   4.5 

Normal   serum    -f    washed  corpuscles   +   streptococci    4.3 

Immune  serum   +   washed   corpuscles   -f-   streptococci    7.3 

Washed  corpuscles  in  NaCl  solution  +  streptococci  1.1 

The  experiment  shows  that  the  opsonin  of  the  immune  serum  is  only 
slightly  disturbed  by  heating  at  60  C.  for  y2  hour,  whereas  that  of  nor- 
mal serum  is  entirely  destroyed  at  that  temperature. 

CONCLUSIONS. 

The  normal  sera  of  man,  rabbits  and  guinea-pigs  have  no  strep- 
tococcidal  power  and  do  not  acquire  such  a  property  in  the  course  of  a 
streptococcus  infection. 

Suspensions  of  organ  cells  of  guinea-pigs  have  no  streptococcidal 
powers. 

Defibrinated  human  blood  is  distinctly  streptococcidal  and  this  prop- 
erty is  roughly  proportional  to  the  number  of  leucocytes  the  blood  con- 
tains per  cubic  millimeter. 

Normal  leucocytes  of  rabbit,  guinea-pig  and  man,  suspended  in 
normal  serum  or  blood,  freely  ingest  non-virulent  streptococci  and  de- 
stroy them.  The  washed  leucocytes  in  NaCl  solution  or  in  heated  serum 
do  not  ingest  these  organisms,  but  the  latter  multiply  in  these  suspen- 
sions. If,  however,  the  streptococci  are  treated  with  normal  serum, 
washed  and  then  added  to  a  suspension  of  washed  leucocytes  they  are 
ingested  and  destroyed  by  the  leucocytes.  In  the  phagocytosis  of  strepto- 
cocci, therefore,  it  is  essential  that  the  organisms  should  first  be  sensi- 
tized, that  is,  acted  upon  by  the  opsonin  of  the  serum. 

The  leucocytes  of  guinea-pigs  and  of  man  take  up  living  strepto- 
cocci in  vivo,  and  in  all  probability  destroy  them.  It  seems  evident, 
therefore,  that  the  phagocytes,  acting  in  conjunction  with  the  opsonin  of 
the  serum,  are  the  most  important  (if  not  the  only)  factors  concerned 


27 

in  combating  streptococcus  infections  in  man  and  in  the  lower  animals. 

Hektoen  and  Ruediger*  have  shown  that  m-8  solutions  of  many  salts 
and  other  substances  inhibit  phagocytosis,  presumably  because  these  sub- 
stances bind  the  opsonin.  I  have  carried  out  a  number  of  experiments 
with  rabbits  for  the  purpose  of  determining  whether  or  not  intravenous 
injections  of  weak  solutions  of  antiseptics  can  prolong  the  animal's  life 
when  inoculated  with  a  fatal  dose  of  streptococcus,  but  have  found  that 
the  animals  thus  treated  invariably  died  earlier  than  the  controls.  We 
must  conclude,  therefore,  that  great  harm  can  be  done  by  the  indiscrimi- 
nate use  of  drugs,  or  antiseptics,  for  the  purpose  of  combating  strepto- 
coccus infections.  The  remedy  used  may  act  on  the  opsonin,  for  in- 
stance, so  as  to  hinder  phagocytosis  and  thus  do  harm  rather  than  good. 

Virulent  streptococci  are  not  freely,  or  scarcely  at  all,  ingested  by 
normal  leucocytes  suspended  in  normal  serum  or  blood,  and  hence  these 
organisms  multiply  in  suspensions  of  leucocytes.  Human  leucocytes  take 
up  virulent  streptococci  somewhat  more  freely  than  do  the  leucocytes 
of  rabbits  and  guinea-pigs. 

Rabbits  can  be  successfully  immunized  with  streptococci  of  mediunr 
virulence,  and  this  immunity  is  clearly  dependent  upon  phagocytosis. 
The  immune  rabbit  serum  has  no  streptococcidal  power,  but  the  leucocytes 
suspended  in  the  immune  blood  or  serum  readily  take  up  and  destroy 
that  strain  of  streptococcus  which  was  used  in  the  immunization.  Other 
strains  of  virulent  streptococci  are  not  taken  up  so  readily  by  leucocytes 
suspended  in  the  immune  serum.  This  is  an  important  fact  to  be  recog- 
nized in  the  serum  therapy  of  streptococcus  infections. 

The  action  of  the  immune  serum  is  not  that  of  a  stimulus  for  the  leu- 
cocytes but  the  effect  is  on  the  cocci.  The  immune  serum  has  acquired 
the  power  to  change  the  cocci  so  that  the  leucocytes  will  ingest  them,  this 
power  being  possessed  only  slightly  by  the  normal  serum. 

The  opsonin  of  human  serum  is  increased  during  the  course  of 
a  streptococcus  infection,  and  according  to  Hektoen  also  after  a  subcuta- 
neous injection  of  heated  streptococci. 

The  immune  opsonin  is  more  resistant  to  heat  than  normal  opsonin. 

*Jonr.  of  Infect.  Dis.,  1905,  II,  128. 


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